Method for production of neuroblasts

ABSTRACT

A method for producing a neuroblast and a cellular composition comprising an enriched population of neuroblast cells is provided. Also disclosed are methods for identifying compositions which affect neuroblasts and for treating a subject with a neuronal disorder, and a culture system for the production and maintenance of neuroblasts.

[0001] This application is a continuation-in-art of application Ser. No.08/001,543 filed on Jan. 6, 1993.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates generally to cell populations derived fromneurons, which are denoted neuroblasts, methods for the production andlong-term in vitro culture of these cell populations, and the use ofneuroblasts in the treatment of various neuronal disorders as well asthe identification of compositions which affect neuroblasts.

[0004] 2. Description of Related Art

[0005] Only a few neuronal cell types have been reported to divide inthe adult brain and adult neurons do not survive well in vitro. To date,even with all of the recent advances in neurobiology, genetics,immunology and molecular biology, no reliable procedure exists toestablish cell lines from the central nervous system (CNS) and neuronaltissues in the absence of immortalization. The generation of clonal celllines from different regions of the brain is important and will greatlyfacilitate the discovery of new neurotrophic factors and theirreceptors, and enhance the understanding of their function.

[0006] The central nervous system contains two major classes of cellsknown as neurons and glial cells. Glial cells include astrocytes,oligodendrocytes and microglia. There are hundreds of different types ofneurons and many different neurotraphic factors which influence theirgrowth and differentiation. Depending on the type of neuron and theregion of the brain in which the neuron resides, a differentneurotrophic factor or specific combination of factors affect thesurvival, proliferation and differentiation of the neuron. Each type ofneuron responds to different combinations of neurotransmitters,neurotrophic factors, and other molecules in its environment

[0007] To date, neuropharmacological studies in the CNS have beendelayed by the lack of cell systems needed to investigate potentiallyuseful neuroactive compounds. In live animals, the complexity of thebrain makes it difficult to effectively measure which cellular receptorsare being targeted by these compounds. Additionally, the expenseinvolved in live animal research and the current controversies stemmingfrom animal rights movements have made in vivo animal studies lessacceptable for initial research. Primary cells from neuronal issue areoften used for CNS studies, however, long-term culture of primaryneurons has not been achieved. Also, only a few attempts to achieve notonly long term culture, but actual proliferation of neuronal cells havebeen reported. In fact, the, proliferation of neuronal cells has provenso elusive that it has become ingrained in the scientific community thatneuronal cells do not proliferate in vitro. As a consequence, freshdissections must be performed for each study in order to obtain thenecessary neuronal cell types, resulting in costly research withincreased variability in the experimental results.

[0008] While some neuronal tumorogenic cells exist they are few innumber and are not well characterized. In general, these tumor celllines do not mimic the biology of the primary neurons from which theywere originally established and, as a result, are not suitable for drugdiscovery screening programs. An vitro primary cultures that would bemore phenotypically representative of primary cells and that couldgenerate continuous cultures of specific neuronal cell lines capable ofproliferation would be invaluable for neurobiological studies and CNSdrug discovery efforts, as well as therapy.

[0009] It has become increasingly apparent that more defined conditionsand further refinements in culture methodology are necessary to produceneuronal cell lines which would enhance the yield of information from invitro studies of the nervous system. Recognition of cell type anddevelopmental stage-specific requirements for maintaining neural cellsin culture as well as the development of a broader range of cultureconditions are required. However, in order to achieve these goals it iscritical to develop optimal culture methods which mimic in vivoconditions which are devoid of the biological fluids used inconventional culture techniques.

[0010] Recently, several researchers have isolated and immortalizedprogenitor cells from various regions of the brain and different stagesof development. Olfactory and cerebellum cells have been immortalizedusing the viral myc (v-myc) ancogene to generate cell lines withneuronal and glial phenotypes (Ryder, et al., J. Neurobiology, 21:356,1990). Similar studies by Snyder, et al. (Cell, 68:33, 1992) resulted inmultipotent neuronal cell lines which were engrafted into the ratcerebellum- to form neurons and glial cells. In other studies, murineneuroepithelial cells were immortalized with a retrovirus vectorcontaining c-myc and were cultured with growth factors to formdifferentiated cell types similar to astrocytes and neurons. (Barlett,et al., Proc.Natl.Acad.Sci.USA, 85:3255,1988).

[0011] Epidermal growth factor (EGF) has been used to induce the invitro proliferation of a small number of cells isolated from thestriatum of the adult mouse brain (Reynolds and Weiss, Science, 255:17071992). Clusters of these cells had antigenic properties ofneuroepithelial stem cells and under appropriate conditions, these cellscould be induced to differentiate into astrocytes and neurons withphenotypes characteristic of the adult striatum in vivo However, itshould be noted that these differentiated neurons were not cultured forlengthy periods of time nor was there any evidence that these cellscould be frozen and then thawed and recultured.

[0012] Cattaneo and McKay (Nature, 347:762, 1990) performed experimentsusing rat striatum to determine the effect of nerve growth factor (NGF)on proliferation of neuronal precursor cells. The cells were dissectedfrom rat embryonic striaturn and exposed to both NGF and basicfibroblast growth factor (bFGF, also known as FGF2). These cells werecultured only nine days in vitro, at which time they had differentiatedinto neurons as determined by assay with neuron-specific markers.

[0013] Neuronal precursor cells from the cerebral hemispheres of 13 dayold rat embryos have been cultured for up to 8 days in the presence ofbFGF at 5 ng/ml (Gensberger, et at., FEBS Lett 217:1, 19874. At thisconcentration, bFGF stimulated only short-term proliferationProliferation and differentiation of primary neurons from both fetal andadult striatum in response to a combination of NGF and bFGF or only EGFhave also been reported (Catteneo, et al., supra; Reynolds and Weiss,supra).

[0014] In view of the foregoing, there is a need for a long-term invitro culture system which would allow large scale production andmaintenance of a neuronal cell population which will proliferate and canbe passaged and subcultured over time. Such homogenous in vitro neuronalcultures will prove invaluable in studying cell populations, theinteractions between these cells and the effects of various neuroactivecompositions on these cells.

SUMMARY OF THE INVENTION

[0015] Recognizing the importance of a system for producing andmaintaining neuronal cells in vitro, the inventors developed a methodand a culture system for producing continuous fetal and adult neuronalcell lines. The development of primary neuronal cultures maintained ascell lines, known as neuroblasts, using neurotrophic factors in theabsence of oncogenic immortalization, now permits investigation offundamental questions regarding the biochemical and cellular propertiesof these cells and the dynamics of interaction between their cellularand chemical environment

[0016] The neuroblasts of the invention can advantageously be used tostably incorporate genetic sequences encoding various receptors, ligandsand neurotransmitters, for example, for use in the treatment of subjectswith neuronal disorders and for identifying compositions which interactwith these molecules.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 shows BrdU staining and NeuroTag™ binding of primaryneurons in culture A. Primary neurons were labeled with BrdU for 1 day;and B. for 4 days C. The neuronal nature of primary cells was determinedby binding with tetanus toxin (NeuroTagr™). Cell bodies and processes ofall cells in culture were stained. Calibration bar=20 μm.

[0018]FIG. 2 illustrates photomicrographs showing the morphologicalchanges that occur during the culture and passaging of primary neurons.A. Primary cell culture after 4 days of plating in N2+bFGF. B. Primarycells 4 days in culture after passage (passage 3). Cells were larger andinterconnected by processes that also increased in size. Smallproliferating cells were visible in the culture. C. Cells passaged(passage 3) and kept in culture for ˜14 days in the presence of bFGF.Negative magnification 33×.

[0019]FIG. 3 shows transmission electron micrographs of primary neuronsin culture. A. A pyramidal-shaped primary hippocampal neuron showingboth the soma and processes, including a major apical process (arrow)and a finer caliber process (arrowhead). Bar=10 μm. B. Enlarged view ofthe neuronal soma shown in panel A. Bar=1 μm. C. A portion of the majorapical process of the neuron shown in panel A. Bar=1 μm. D. Contactbetween two neuritic processes. Bar=0.1 μm.

[0020]FIG. 4 shows scanning electron micrographs of primary neurons inculture. A. Overview of primary hippocampal neurons in culture includingwell-differentiated pyramidal somata (arrow) with large processescontaining multiple levels of branching and less-differentiated, roundedneurons with large, extended processes (arrowheads). Bar=50 μm B. Amajor apical dendrite emerging from a well-differentiated pyramidalneuron showing a smooth, regular caliber process just proximal to thefirst (major) bifurcation with several smaller processes, possibly axonsemerging from it. The PORN/laminin coating the vessel surface can beseen as a porous carpeting which is absent in some patches. Bar=2 m C. Awell-differentiated neuron (in the middle of the field) possessing alarge pyramidal soma (compare to FIG. 3A) and a large apical dendrite(arrowheads) contacted by a number of processes from other neurons.Other less-differentiated neurons which are fixed in the process ofdividing were also present (arrows). Bar=20 μm. D. Enlarged view of thedividing neuron in the upper field of view in panel C. Bar=10 μm.

DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention provides an in vitro method for producingan isolated neuronal cell population. These cells, termed neuroblastscan be produced by utilizing methodology which comprises culturing aneuronal cell in a serum-free media supplemented with at least onetrophic factor using a vessel which allows attachment of the cell. Thismethod allows the generation of continuous, neuronal cell cultures fromdifferent regions of the brain, from both fetal and adult tissue, whichare capable of proliferation.

[0022] The invention also provides a method of identifying compositionswhich affect a neuroblast, such as by inhibiting or stimulating theneuroblast proliferation. A culture system useful for the production andmaintenance of a neuroblast comprising a serum-free basal mediacontaining at least one trophic factor and a vessel which allowsattachment of the neuroblast is also provided. An enriched population ofneuroblast cells produced by the method of the invention is alsoprovided and can be further utilized for the treatment of a subject witha neuronal cell disorder or alternatively, to screen compositions whichaffect the neuroblast.

[0023] As used herein, the term “neuroblast” refers to a non-glial cellof neuronal lineage which has been perpetualized. Neuronal“perpetualization” refers to the procedure whereby a non-glial cell ofthe neuronal lineage is treated with growth factors such that it iscapable of indefinite maintenance, growth and proliferation in vitro.Typically, a primary culture, one in which the tissue is removed from ananimal, is placed in a culture vessel in appropriate fluid medium, andhas a finite lifetime. In contrast, continuous cell lines proliferateand thus can be subcultured, i.e., passaged repeatedly into new culturevessels. Continuous cell lines can also be stored for long periods oftime in a frozen state in the vapor phase of liquid nitrogen when acryopreservatve is present, e.g., 10% dimethylsulfoxide or glycerol. Theneuroblast of the invention can be maintained in long-term culture as acell line closely resembling primary cultures, but without resort tooncogenic immortalization. Rather, “perpetualization” establishes acontinuous culture from a primary neuronal cell by utilizing a specificgrowth factor or combination of growth factors. This perpetualizationtechnique is novel in that no gene transfer or genetic manipulation isrequired and, as a consequence, the cells more closely resemble primarycultures.

[0024] There are hundreds of different types of neurons, each withdistinct properties. Each type of neuron produces and responds todifferent combinations of neurotransmitters and neurotrophic factors.Neurons do not divide in the adult brain, nor do they generally survivelong in vitro. The method of the invention provides for the isolationand growth of perpetualized neurons, or neuroblasts, in vitro; fromvirtually any region of the brain and spinal cord. Either embryonic oradult neurons can be utilized for the development of neuroblast celllines. The neuronal cell of the invention, which is utilized forproduction of a neuroblast, can be derived from any fetal or adultneural tissue, including tissue from the hippocampus, cerebellum, spinalcord, cortex (e.g., motor or somatosensory cortex), striatum, basalforebrain (cholenergic neurons), ventral mesencephalon (cells of thesubstantia nigra), and the locus ceruleus (neuroadrenaline cells of thecentral nervous system).

[0025] The liquid media for production of a neuroblast of the inventionis supplemented with at least one trophic factor to support the growthand proliferation of a neuroblast Trophic factors are molecules whichare involved in the development and survival of neurons. They are oftensynthesized in the brain, have specific receptors, and influence thesurvival and function of a subset of neurons. Examples of such factorsinclude nerve growth factor (NGF), brain-derived neurotrophic factor(BDNF), neurotrophin-3, -4, and -5 (NTF-3, -4, -5), ciliary neurotrophicfactor (CNTF), basic fibroblast growth factor (bFGF), acidic fibroblastgrowth factor (aFGF), platelet derived growth factor (PDGF), epidermalgrowth factor (EGF), insulin-like growth factor-I and -II (IGF-I, -II),transforming growth factor (TGF) and lymphocyte infiltratingfactor/cholinergic differentiating factor (LIF/CDF). The specificity andselectivity of a trophic factor are determined by its receptor.Preferably, the trophic factor utilized in the invention is aneurotrophic factor. Preferably, the neurotrophic factor added to thebasal media for production of a neuroblast according to the method ofthe invention is bFGF. The neurotrophic factor which allows growth andproliferation of the neuroblast in vitro will depend on the tissueorigin of the neuroblast. However, for most neuronal cells, bFGF will bethe preferred neurotrophic factor.

[0026] The vessel utilized for production of a neuroblast must provide asurface which allows attachment of the neuronal cell. Such vessels arealso preferred once the isolated neuroblast culture has been produced.The surface used to enhance attachment of the neuronal cell can be theactual inner layer of the vessel or more indirectly, the surface of asupplemental insert or membrane which resides within the vessel.Attachment may be accomplished by any means which allows the cell togrow as a monolayer on a vessel. Attachment enhancing surfaces can beproduced directly, such as by advantageous selecting of appropriateplastic polymers for the vessel or, indirectly, as by treating thesurface in the vessel by a secondary chemical treatment Therefore,“attachment” refers to the ability of a cell to adhere to a surface in atissue culture vessel, wherein the attachment promoting surface is indirect contact with neuronal cells, which otherwise would grow in athree-dimensional cellular aggregate in suspension. Attachment, oradherence, of a neuronal cell to the vessel surface allows it to beperpetualized.

[0027] In addition to interactions with soluble factors, most cells invivo, including neuronal cells, are in contact with an extracellularmatrix, a complex arrangement of interactive protein and polysaccharidemolecules which are secreted locally and assemble into an intricatenetwork in the spaces between cells. Therefore, the addition of anextracellular matrix protein to the surface of the culture vessel formsan insoluble matrix which allows neuronal cells in culture to adhere ina manner which closely corresponds to the in vivo extracellular matrixThe neuroblast of the invention can be preferably produced by coatingthe surface of a vessel, such as a tissue culture dish or flask, with apolybasic amino acid composition to allow initial attachment. Suchcompositions are well known in the art and include polyornithine andpolylysine. Most preferably, the polybasic amino acid of the inventionis polyornithine. Additionally, the surface of the vessel may be coatedwith a known extracellular matrix protein composition to enhance theneuroblast's ability to grow and form processes on the substrate. Suchcompositions include laminin, collagen and fibronectin. Otherextracellular matrix proteins that can be used in conjunction with apolybasic amino acid will be apparent to one of skill in the art.Additionally, for the production of adult neuroblasts, it is preferableto initially culture the cells in the presence of serum.

[0028] The neuroblast of the invention is useful as a screening tool forneuropharmacological compounds which affect a biological function of theneuroblast. Thus, in another embodiment, the invention provides a methodfor identifying a composition which affects a neuroblast comprisingincubating the components, which include the composition to be testedand the neuroblast, under conditions sufficient to allow the componentsto interact, then subsequently measuring the effect the composition onthe neuroblast The observed effect on the neuroblast may be eitherinhibitory or stimulatory. For example, a neuroactive compound whichmimics a neurotransmitter or binds to a receptor and exhibits either anantagonistic or agonist effect, thereby inhibiting or stimulating abiological response in the neuroblast, can be identified using themethod of the invention. The occurrence of a biological response can bemonitored using standard techniques known to those skilled in the art.For example, inhibition or stimulation of a biological response may beidentified by the level of expression of certain genes in theneuroblast. Such genes may include early response genes such as fos, mycor jun (Greenberg, M. and Ziff, E. Nature, 311:433, 1984; eds. Burck, etal., in Oncogenes, 1988, Springer-Verlag, New York.). Other genes,including those which encode cell surface markers can also be used asindicators of the effects neuropharmacological compounds on theneuroblasts of the invention Methods for measurement of such effectsinclude Northern blot analysis of RNA (transcription), SDS-PAGE analysisof protein (translation), [C³H]-thymidine uptake (DNA synthesis) andantibody reactivity (both intracellular and extracellular). Othercommonly used methods will be apparent to those of skill in the art.]

[0029] Neuroactive drugs which act similarly to those already known toaffect neuronal cells can thus be identified. For example, new drugsthat alleviate anxiety, analogously to Valium, which augment orstimulate the action of the important inhibitory transmittergamma-aminobutyric acid (GABA), can be identified. Antidepressants, suchas Prozac, enhance the action of serotonin, an indoleamine with a widevariety of functions. Other drugs can be readily identified using theneuroblasts according to the method of the invention. Other examplesinclude psychoactive compounds. For example, cocaine facilitates theaction of dopamine, whereas certain antipsychotics antagonize or inhibitthis catecholamine. Another example is nicotine which activates theacetylcholine receptors which are distributed throughout the cerebralcortex. Therefore, by using neuroblasts derived from neuronal cells fromthe appropriate regions of the brain, drugs and trophic factors whichbind various receptors and would produce similar effects on neuronalcells can be identified using the method of the invention.

[0030] As described above, perpetualization of a neuronal cell can be,accomplished without the use of oncogenic intervention. However, ifdesired the neuroblast of the invention may be immortalized to maintainthe cell at a defined developmental stage. The present techniques forimmortalization typically involve the transfection of an oncogene to thecell, therefore, immortalization of a neuroblast can be achieved byintroduction of at least one oncogene to the neuroblast. Transfection ofthe oncogene can be accomplished by several conventional methods wellknown to those skilled in the art, including using recombinantretroviruses, chemical, or physical methods. Recombinant retrovirustransfer is the preferred method of the invention for immortalization ofneuroblasts.

[0031] The host neuroblast can be immortalized with a particularonco'gene by such methods of transfection as calcium phosphateco-precipitation, conventional mechanical procedures such asmicroinjection, insertion of a plasmid encased in liposomes, or by useof viral vectors. For example, one method is to use a eukaryotic viralvector, such as simian virus 40 (SV40) or bovine papilloma virus, totransiently infect or transform the neuroblast (Eukaryotic ViralVectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).

[0032] Various viral vectors which can be utilized for immortalizationas taught herein include adenovirus, herpes virus, vaccinia, andpreferably, an RNA virus such as a retrovirus. Preferably, theretroviral vector is a derivative of a murine or avian retrovirus.Examples of retroviral vectors in which a single foreign gene can beinserted include, but are not limited to: Moloney murine leukemia virus(MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumorvirus (MuMTV), and Rous Sarcoma Virus (RSV). A number of additionalretroviral vectors can incorporate multiple genes. All of these vectorscan transfer or incorporate a gene for a selectable marker so thattransduced cells can be identified and generated.

[0033] Since recombinant retroviruses are defective, they requireassistance in order to produce infectious vector particles. Thisassistance can be provided, for example, by using helper cell lines thatcontain plasmids encoding all of the structural genes of the retrovirus(gag, env, and pol genes) under the control of regulatory sequenceswithin the long terminal repeat (LTR). These plasmids are missing anucleotide sequence which enables the packaging mechanism to recognizean RNA transcript for encapsidation. Helper cell lines which havedeletions of the packaging signal include, but are not limited to ψ2,PA317, PA12, CRIP and CRE, for example. These cell lines produce emptyvirions, since no genome is packaged. If a retroviral vector isintroduced into such cells in which the packaging signal is intact, butthe structural genes are replaced by other genes of interest, the vectorcan be packaged and vector virion produced. The vector virions producedby this method can then be used to infect a tissue cell line, such asNIH 3T3 cells, to produce large quantities of chimeric retroviralvirions.

[0034] Alternatively, NIH 3T3 or other issue culture cells can bedirectly transfected with plasmids encoding the retroviral structuralgenes gag, pol and env, by conventional calcium phosphate or lipofectiontransfection. These cells are then transfected with the vector plasmidcontaining the genes of interest. The resulting cells release theretroviral vector into the culture medium.

[0035] Herpes virus-based vectors may also be used to transfer genesinto a neuroblast. Since herpes viruses are capable of establishing alatent infection and an apparently non-pathogenic relationship with someneural cells, such vector based on HSV-1, for example, may be used.Similarity, it should be possible to take advantage other human andanimal viruses that infect cells of the CNS efficiently, such as rabiesvirus, measles, and other paramyxoviruses and even the humanimmunodeficiency retrovirus (HIV), to develop useful delivery andexpression vectors.

[0036] When a recombinant retrovirus is engineered to contain animmortalizing oncogene, the oncogene can be any one of those known toimmortalize. For example, such commonly used immortalizing genes includegenes of the myc family (both c-myc and v-myc) (Bartlett, et al.,Proc.Natl.Acad.Sci.USA 85:3255, 1988), adenovirus genes (E1a 12s and E1a13s) (Ruley, et al., Nature 304:602, 1983), the polyoma large T antigenand SV40 large T antigen (Frederiksen, et al., Neuron 1:439, 1988).Preferably, the oncogene used to immortalize the neuroblast of theinvention is v-myc. Other genes, for example other nuclear oncogenes,that immortalize a cell but may require a second gene for completetransformation, will be known to those of skill in the art.

[0037] The same transfection methods described above for immortalizationof a neuroblast can be utilized to transfer other exogenous genes to theneuroblast of the invention. An “exogenous gene” refers to geneticmaterial from outside the neuroblast which is introduced into theneuroblast. An example of a desirable exogenous gene which would beuseful for the method of identifying neuropharmacological compounds is agene for a receptor molecule. For example, such neuronal receptorsinclude the receptor which binds dopamine, GABA, adrenaline,noradrenaline, serotonin, glutamate, acetylcholine and various otherneuropeptides. Transfer and expression of a particular receptor in aneuroblast of specific neural origin, would allow identification ofneuroactive drugs and trophic factors which may be useful for thetreatment of diseases involving that neuroblast cell type and thatreceptor. For example, a neuroactive compound which mimics aneurotransmitter and binds to a receptor and exhibits either anantagonistic or agonist effect, thereby inhibiting or stimulating aresponse in the neuroblast, can be identified using the method of theinvention.

[0038] In another embodiment, the invention provides a culture systemuseful for the production and maintenance of a neuroblast comprising aserum-free basal media containing at least one trophic factor and avessel having a surface which allows attachment of the neuroblast. Theculture system can be utilized to produce a neuroblast from any tissueof neural origin as described above. The “serum-free basal media” of theinvention refers to a solution which allows the production andmaintenance of a neuroblast. The basal media is preferably a commonlyused liquid tissue culture media, however, it is free of serum andsupplemented with various defined components which allow the neuroblastto proliferate. Basal media useful in the culture system of theinvention is any tissue culture media well known in the art, such asDulbecco's minimal essential media, which contains appropriate aminoacids, vitamins, inorganic salts, a buffering agent, and an energysource. Purified molecules, which include hormones, growth factors,transport proteins, trace elements, vitamins, and substratum-modifyingfactors are added to the basal media to replace biological fluids. Forexample, progesterone, sodium selenite, putrescine, insulin andtransferrin are typically added to the basal media to enhance neuroblastgrowth and proliferation. For the culture system of the invention, onlytwo of the defined supplements are necessary to sustain growth ofneurons alone (transferrin and insulin), whereas the combination of thefive supplements above have a highly synergistic growth-stimulatingeffect Deletion of any single supplement results in markedly diminishedgrowth of the neuroblast. An example of a preferred prototype mediumwhich contains these elements is. N2 medium (Bottenstein and Sato, etal., Proc.Natl.Acad.Sci.USA, 76:514, 1979). The optimal concentration ofthe supplements are as follows: 5 μg/ml insulin, 100 μg/ml transferrin,20 nM progesterone, 100 μM putrescine, and 30 nM selenium (as Na₂SeO₃).

[0039] The basal media of the culture system further contains at leastone trophic factor for the production and maintenance of a neuroblast.Most preferably, neurotrophic factors are utilized and specificallybFGF. bFGF is utilized in the basal media at a concentration from about1 ng/ml to about 100 ng/ml, more specifically from about, 5 ng/ml toabout 70 ng/ml, and most preferably from about 15 ng/ml to about 60ng/ml. Neural cultures are generally maintained at pH 72-7.6. A higherrequirement for glucose is also necessary for neural as opposed tonon-neural cells. Therefore, the basal media of the invention contains aconcentration of from about 0.01% to about 1.0% glucose and preferablyfrom about 0.1% to about 0.6% glucose.

[0040] The invention also provides a cellular composition comprising anenriched population of neuroblast cells. The composition preferablycontains a majority of or at least about 90% neuroblasts. The neuroblastcells are derived from any CNS neural tissue such as from any region ofthe brain, as described above, or from the spinal cord. The neuroblastmay be further immortalized with an oncogene, or it may contain anexogenous gene encoding a receptor or a ligand for a receptor.

[0041] The present invention also provides a method of treating asubject with a neuronal cell disorder which comprises administering tothe subject a therapeutically effective amount of the neuroblast of theinvention. “Therapeutically effective” as used herein, refers to thatamount of neuroblast that is of sufficient quantity to ameliorate thecause of the neuronal disorder. “Ameliorate” refers to a lessening ofthe detrimental effect of the neuronal disorder in the patient receivingthe therapy. The subject of the invention is preferably a human,however, it can be envisioned that any animal with a neuronal disordercan be treated with the neuroblast of the invention. Preferably, theneuroblast is derived from neuronal tissue of the same species as thespecies of the subject receiving therapy.

[0042] The method of treating a subject with a neuronal disorder entailsintracerebral grafting of neuroblasts to the region of the CNS havingthe disorder. Where necessary, the neuroblast can be geneticallyengineered to contain an exogenous gene. The disorder may be from eitherdisease or trauma (injury). Neuroblast transplantation, or “grafting”involves transplantation of cells into the central nervous system orinto the ventricular cavities or subdurally onto the surface of a hostbrain. Such methods for grafting will be known to those skilled in theart and are described in Neural Grafting in the Mammalian CNS, Bjorklundand Stenevi, eds., (1985), incorporated by reference herein. Proceduresinclude intraparenchymal transplantation, (i.e., within the host brain)achieved by injection or deposition of tissue within the host brain soas to be apposed to the brain parenchyma at the time of transplantation.

[0043] Administration of the neuroblasts of the invention into selectedregions of the recipient subject's brain may be made by drilling a holeand piercing the dura to permit the needle of a microsyringe to beinserted. The neuroblasts can alternatively be injected intrathecallyinto the spinal cord region. The neuroblast preparation of the inventionpermits grafting of neuroblasts to any predetermined site in the brainor spinal cord, and allows multiple grafting simultaneously in severaldifferent sites using the same cell suspension and permits mixtures ofcells from different anatomical regions. The present invention providesa method for transplanting various neural tissues, by providingpreviously unavailable proliferating neuroblasts and a culture systemfor production of these neuroblas's in order to grow a sufficient numberof cells for in vitro gene transfer followed by in vivo implantation.

[0044] The neuroblast used for treatment of a neuronal disorder mayoptionally contain an exogenous gene, for example, an oncogene, a genewhich encodes a receptor, or a gene which encodes a ligand. Suchreceptors include receptors which respond to dopamine, GABA, adrenaline,noradrenaline, serotonin, glutamate, acetylcholine and otherneuropeptides, as described above. Examples of ligands which may providea therapeutic effect in a neuronal disorder include dopamine,adrenaline, noradrenaline, acetylcholine, gamma-aminobutyric acid andserotonin. The diffusion and uptake of a required ligand after secretionby a donor neuroblast would be beneficial in a disorder where thesubject's neural cell is defective in the production of such a geneproduct. A neuroblast genetically modified to secrete a neurotrophicfactor, such as nerve growth factor, (NGF), might be used to preventdegeneration of cholinergic neurons that might otherwise die withouttreatment. Alternatively, neuroblasts to be grafted into a subject witha disorder of the basal ganglia, such as Parkinson's disease, can bemodified to contain an exogenous gene encoding L-DOPA, the precursor todopamine. Parkinson's disease is characterized by a loss of dopamineneurons in the substantia-nigra of the midbrain, which have the basalganglia as their major target organ. Alternatively, neuroblasts derivedfrom substantia-nigra neuronal cells which produce dopamine could beintroduced into a Parkinson's patient brain to provide cells which“naturally” produce dopamine.

[0045] Other neuronal disorders that can be treated similarly by themethod of the invention include Alzheimer's disease, Huntington'sdisease, neuronal damage due to stroke, and damage in the spinal cord.Alzheimer's disease is characterized by degeneration of the cholinergicneurons of the basal forebrain. The neurotransmitter for these neuronsis acetylcholine, which is necessary for their survival. Engraftment ofcholinergic neuroblasts, or neuroblasts containing an exogenous gene fora factor which would promote survival of these neurons can beaccomplished by the method of the invention, as described. Following astroke, there is selective loss of cells in the CA1 of the hippocampusas well as cortical cell loss which may underlie cognitive function andmemory loss in these patients. Once identified, molecules responsiblefor CA1 cell death can be inhibited by the methods of this invention.For example, antisense sequences, or a gene encoding an antagonist canbe transferred to a neuroblast and implanted into the hippocampal regionof the brain.

[0046] The method of treating a subject with a neuronal disorder alsocontemplates the grafting of neuroblasts in combination with othertherapeutic procedures useful in the treatment of disorders of the CNS.For example, the neuroblasts can be co-administered with agents such asgrowth factors, gangliosides, antibiotics, neurotransmitters,neurohormones, toxins, neurite promoting molecules and antimetabolitesand precursors of these molecules such as the precursor of dopamine,L-DOPA.

[0047] The following examples are intended to illustrate but not limitthe invention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively be used.

EXAMPLES

[0048] The following examples show neuronal proliferation ofhippocampal, spinal cord, substania nigra, basal forebrain, and otherneuronal tissue cells from fetal rats cultured over 5 months with bFGF.In addition, adult hippocampus was cultured with bFGF in a defined mediafor more than 7 months. The examples also provide methodology for thegeneration, differentiation and long term culture of numerous cell typesfrom fetal and adult neuronal tissue and describe the morphological,immunocytochemical, ultrastructural and molecular characteristics ofproliferating non-neuronal and neuronal cell types in the adult bFGFtreated cultures.

[0049] Proliferating cells that incorporated bromodeoxyuridine wereimmunopositive for neuron-specific enolase. Cells with polarizedmorphologies typical of well-differentiated neurons were immunopositivefor the high molecular weight subunit of neurofilament protein (NFh),characteristic of mature neurons, the middle and low subunits ofneurofilament protein and microtubule-associated protein 2 (MAP-2).Cells from adult mammalian hippocampus were capable of proliferation aswell as long-term neurogenesis and neuronal differentiation in vitro.These cells may be a source of replacement cells in neuronal grafting.Further, the induction of proliferation and differentiation of thesecells in vivo would be useful for replacement or augmentation ofneuronal loss or degeneration.

Example 1 Materials and Methods

[0050] Materials: DMEM:F12 medium, N2 supplement and laminin wereobtained from Gibco/BRL (Bethesda, Mo.); polyomithine (PORN) wasobtained from Sigma (St Louis, Mo.). Recombinant bFGF was fromSyntex/Synergen Consortium (Boulder, Colo.). Bovine bFGF was purchasedfrom R&D, Minneapolis, Minn. NeuroTag™ green was obtained fromBoehringer Mannheim, Indianapolis, Ind. Cell proliferation kitcontaining bromodeoxyuridine (BrdU), anti-BrdU antibody andstreptavidin/Texas Red was purchased from Amersham. Arlington Heights,Ind. The antibodies used to determine the phenotypes of cells in culturewere obtained from the following sources and used at the indicateddilutions: polyclonal rabbit antifleurofilament 200 (NF) (1:500;Chemicon International, Temecula, Calif.), monoclonal anti-neuronspecific enolase (NSE) (1:50; DAKO, Carpenteria, Calif.), monoclonalanti-glia fibrillary acidic protein (GFAP) (1:500-1:10,000; Amersham,Arlington Heights, Ill.), monoclonal anti-vimentin (1:800; BoehringerMannheim, Indianapolis, Ind.), monoclonal anti-OX-42 (1:5000; Serotec,Indianapolis, Ind.), polyclonal anti-galactocerebroside (Gal C) (1:5000;Advanced Immunochemical Services, Long Beach, Calif.), monoclonalanti-microtubule associated protein (MAP 2) (1:500; SigmaImmunochemicals, St. Louis, Mo.), polyclonal anti-fibronectin (1:2000;Telios, La Jolla, Calif.). Polyclonal nestin antibody (1:15,000) wasfrom Dr. R. McKay, MIT, Cambridge, Mass., and monoclonal high affinitybFGF receptor antibody (1:20) was from Dr. A. Baird, Whittier Institute,La Jolla, Calif. Polyclonal anti-GFAP (1:2000) was from Dr. L F. Eng,Stanford University, Palo Alto, Calif.

[0051] Cell Culture. The brains of Fisher 344 rats (E16) were dissected,the meninges were removed and the hippocampi were isolated. Hippocampiwere transferred to a 15 ml tissue culture tube and the volume wasadjusted to 1-2 ml with phosphate-buffered saline (pH 7.4) supplementedwith 0.6% glucose (PBS-G). Hippocampi were mechanically dissociated bytrituration with a pasteur pipet (˜20×) followed by trituration with apasteur pipet fire-polished to significantly reduce the pipet bore(˜20×). The cell suspension was pelleted by centrifugation at 1000 rpmfor 5 minutes at room temperature. Cells were taken up in ˜20 ml N2medium (1:1 mixture of DMEM:F12 containing 20 nM progesterone, 30,nMsodium selenite, 100 μM putrescine, 3.9 mM glutamine, 5 μM/ml insulin,100 μg/ml transferrin) and the cell number was quantified with ahemocytometer. Tissue culture plates were coated with polyomithine(PORN; 10 μg/ml) followed by laminin (10 μg/ml). Approximately0.5-10×10⁶ cells/well were plated on PORN/laminin-coated 6 well platesin N2 medium containing 20 ng/ml bFGF (N2+bFGF) and cultured at 37° C.in 5% CO₂. Medium was changed every 3-4 days with fresh N2+bFGF. Forpassaging, cells were trypsinized (ATV trypsin, Irvine Scientific, SantaAna, Calif.) and then taken up in N2+bFGF. Cells were pelleted bycentrifugation and supernatant containing trypsin was removed. Cellswere resuspended in 10 ml N2÷bFGF and plated. Cells could be frozen inliquid nitrogen in N2+bFGF+10% dimethylsulfoxide (DMSO). For culturing,cells were thawed quickly at 37° C., added to 10 ml N2+bFGF, centrifugedto remove DMSO, resuspended in fresh N2+bFGF and plated as describedbefore.

[0052] BrdU Incorporation Experiments. Primary neurons (passage 5) weregrown for 3 days, whereupon the media was changed. On the following daycells were incubated with BrdU for either 1 day or 4 days. Cells werefixed, washed and then treated with a monoclonal antibody against BrdUfor 1 hour After washing, cells were reacted with biotinylatedanti-mouse antibody (Vector Laboratories, Burlingame, Calif.) followedby streptavidin/Texas Red complex. Stained cultures were examined with aBioRad MRC600 confocal scanning laser microscope equipped with akrypton-argon laser using the YHS filter set (568 EX, 585 LP). Confocalfluorescent and Nomarski transmitted collected images were transferredto an Apple Macintosh Quadra 700, merged using Adobe Photoshop 2.01, andprinted out on a GCC film recorder.

[0053] Neurotag™ Binding. Primary neurons (passage 5) grown in culturefor 6 days were incubated with 10 μg/ml recombinant tetanus toxin Cfragment conjugated to fluorescein isothiocyanate (NeuroTag™) in N2+bFGFand bovine serum albumin (0.1 mg/ml) for 2 hours. After washing thecells were examined in a BioRad confocal microscope as described forBrdU stained cells except using the BHS filter (488 ED, 515 LP).

[0054] Immunohistochemistry. Cells were passaged (passage 3; 4 days inculture after plating), grown in a 24-well plate, fled in 4%paraformaldehyde in PBS, and then permeabilized with 0.25% Triton X-100in Tris buffered saline. Cells were incubated overnight at 4° C. withpolyclonal or monoclonal antibodies in the presence of 1% normal horseserum (for monoclonal antibody) or 10% normal goat serum (for polyclonalantibody). After washing, cells were incubated with biotin conjugatedgoat anti-rabbit IgG or horse anti-mouse IgG antibodies (VectorLaboratories, Burlingame, Calif.) for 1 hour at room temperature,followed by incubation for 1 hour at room temperature with a pre-formedmixture of avidin-biotinylated horseradish peroxidase complex(Vectastain Elite ABC kit). The reaction products were visualized withdiaminobenzidine (DAB) histochemistry.

[0055] Transmission Electron Microscopy (TEM). Cultures (passage 3; fourdays after plating) grown on LabTek™ permanox slides (Ted Pella, Inc.,Redding, Calif.) were fixed in 2% glutaraldehyde in 100 mM PO₄ at 37° C.for 2 hours and then rinsed and postfixed in 1% aqueous OSO₄ for 1 hourat room temperature. Cultures were then dehydrated in a graded ethanolseries, infiltrated with Araldite resin and polymerized in situ. Theglass slide was separated from the polymerized resin from which blocksof cultured cells were cut and glued to resin blanks. Sections were cutparallel to the culture substrate at a thickness of 70 nm. Sectionscollected on 300 mesh copper grids were stained with uranyl acetate andlead citrate and examined with a Phillips CM10 transmission electronmicroscope at 80 kV.

[0056] Scanning Electronic Microscopy (SEM). Cultures (passage 2; fourdays after plating) grown on LabTek™ glass slides were prepared as forTEM up through ethanolic dehydration. The plastic chambers were thenremoved, leaving the sealing gasket in place, and the slide was placedinto a Pelco critical point dryer. Following drying, the slide wascoated with gold-palladium to a thickness of 300 Å in a Technics sputtercoater. The cells were examined in a Cambridge Stereoscan 360 scanningelectron microscope at 10 kV.

[0057] Gene Transfer into Neurons. Approximately 1×10⁶ producer cellswere plated on PORN/laminin coated wells in a 6 well plate and grownovernight at 37° C. in 5% CO₂. Virus from producer cells was collectedafter overnight incubation in DMEM (Dulbecco's minimum essential medium)containing 5% fetal calf serum (FCS) or 5% bovine calf serum (BCS).Virus containing media was filtered through 0.45 μm filters and thenmixed with polybrene (8 μg/ml) and bFGF (20 ng/ml). Media was removedfrom neuronal cultures and virus containing media was added to neuronalcultures and incubated overnight at 37° C. in 5% CO₂. After thisinfection period, the media was removed and replaced with N2 mediacontaining 20 ng/ml bFGF. When the expression vector contained theneomycin resistant gene, the infected cells were selected in thepresence of G418 (400 μg/ml). Cells were passaged and maintained asdescribed above.

[0058] The expression vectors and producer cells used were as follows:

[0059] 1. Avian v-myc gene was expressed from MLV-LTR promoter andbacterial neomycin resistant gene was expressed from thymidine kinase(TK) promoter (Ryder, et al., J. Neurobiol., 21:356375, 1989; Kaplan, etal., J. Virol, 61:1731-1.734, 1987; and Land, et al., Mol. Cell. Biol.,6:1917-1925, 1986). A producer line was generated from ψ2 cells. Thesecells grew in DMEM containing 10%. FCS and 400 μg/ml G418. The daybefore the infection, the medium was changed with fresh DMEM containing5% FCC.

[0060] 2. Bacterial β-galactosidase gene was expressed from the MLV-LTRpromoter. This expression vector contains a part of the gag gene andproduces very high titer virus. There is no neomycin resistant gene inthis vector. This vector was from Dr. Richard Mulligan, MIT, Cambridge,Mass.

[0061] A promoter line was generated from CRIP cells. These cells growin DMEM containing 10% BOB. The day before the infection, the medium waschanged with DMEM containing 5% BCS.

Example 2 Growth of Neurons In Vitro

[0062] The chemically defined medium, N2 (Bottenstein and Sato, Proc.Natl. Acad. Sci. USA, 76: 514-517, 1980; Bottenstein, J. E, In: Cellculture in the neurosciences, J. E Bottenstein and G. H. Sato, Eds.,Plenum Press, New York, N.Y., pp 3-43, 1985; di Porizo, et al., Nature,288:370-373, 1980), has been used to reproducibly generate short-termvirtually pure neuronal cultures (Bottenstein, et al., Exp. Cell Res.,125:183-190, 1980; Barnes and Sato, Anal. Biochem., 102:255-302, 1980)This medium does not support the survival or proliferation ofnon-neuronal cells and it is possible to obtain >95% pure neuronalculture. In defined medium, primary cultures of hippocampal neurons diewithin 7 days but can be maintained for 24 weeks in the presence ofhippocampal explants, a feeder layer of astrocytes, inastrocyte-conditioned medium (Banker, G. A., Science, 209:809-610, 1980)or in the presence of bFGF (Walicke and Baird, Proc. Natl. Acad. Sci.USA, 83:3012-3016, 1986; Walicke; P. A.; J. Neurosci., 8:2618-2627,1988; Walicke, et al., In: Prog. Brain Res., vol. 78, D. m Gash and J.R. Sladek, Eds. (Elsevier Science Publishers B.V.), pp 333-338, 1988).However, cells continued to die slowly and few cells remained after 1month (Walicke, P., et al., Proc. Natl. Acad. Sci. USA, 83:3012-3016,1986).

[0063] bFGF at 20 ng/ml, a concentration of about 100 fold higherconcentration than that used before to study the survival and elongationof axons (Walicke, P. et al., Proc. Natl. Acad. Sci. USA, 83:3012-3016,1986; Walicke, P. A., J. Neurosci., 8:2618-2627, 1988), showed dramaticproliferative effects on hippocampal cells. This proliferative propertyof bFGF was used to promote continued proliferation of primaryhippocampal cells to form a long-term culture. Cells cultured in 20ng/ml bFGF began proliferating by 2 days, with a doubling time of 4days. Primary cells became contact inhibited for growth and reached aplateau after day 7, although growth continued within aggregates (FIG.2C).

[0064] To test whether division was occurring in all cells or only in asubpopulation, cultures were incubated with BrdU for 1 or 4 days and thelabeled nuclei were visualized by indirect immunofluorescence using ananti-BrdU-antibody (FIGS. 1A, B).

[0065]FIG. 1 shows BrdU staining and NeuroTag™ binding of primaryneurons in culture. Primary neurons were labeled with BrdU for 1 day (A)and for 4 days (B). Only a few cells were stained on day 1, but by day 4all cells were stained, indicating that all cells in the culture wereproliferating. The neuronal nature of primary cells was determined bybinding with tetanus toxin (NeuroTag™) (C). Cell bodies and processes ofall cells in culture were stained. Calibration bar=20 μm. After day 1,the nuclei of only a small fraction of cells were immunostained (FIG.1A) but almost the entire cell population was immunostained after 4 daysof incubation with BrdU (FIG. 1B).

[0066] To establish long-term cultures, cells were trypsinized andpassaged. The passaged cells (up to 6 passages tested) grew as well asthe original culture did Cells were frozen in liquid nitrogen, thawedand cultured again. When cells at different passage numbers were thawedand re-cultured, they grew equally well regardless of the passagenumber. Freeze-thawed cells showed the same morphology as the cells keptcontinuously in culture.

[0067] Other cells derived from neuronal tissue have also been studiedfor their ability to grow and be maintained in N2 media in the presenceof bFGF. Table 1 shows the optimum concentrations of bFGF for culture ofthe various cell lines. TABLE 1 REGION OF CNS CONCENTRATION OF bFGF(np/ml)* Hippocampus 20 Septum 100 Striatum 20 Cortex 20 Locus Coeruleus50 Ventral Mesencephalon 50 Cerebellum 20 Spinal Cord 20

Example 3 Characterization of Cells

[0068] Several independent criteria were used to show tat the cells inthe cultures were indeed neurons. These included their morphologicalcharacteristics during growth, expression of neuronal markers andultrastructural analysis by transmission and scanning electronmicroscopy.

[0069] Cell morphology in culture was similar to that described forshort-term cultures of neurons (Banker and Cowan, Brain Res.,126:397425, 1977; Banker and Cowan, J. Comp. Neurol., 187:469-494, 1979)(FIGS. 2A, B, C). FIG. 2 illustrates photomicrographs showing themorphological changes that occur during the culture and passaging ofprimary neurons. A shows primary cell culture after 4 days of plating inN2÷bFGF contained numerous proliferating and process-bearing cells. Bshows' primary cells 4 days in culture after passage (passage 3). Cellswere larger and interconnected by processes that also increased in size.Small proliferating cells were visible in the culture. C shows cellspassaged (passage 3) and kept in culture for ˜14 days in the presence ofbFGF formed aggregates and were interconnected by an extensive networkof processes forming a lattice-type pattern (Negative magnification33×).

[0070] Cells were immunostained for several different antigenic markers.Cells were stained with anti-NF (200 KD) antibody (D); with anti-NSEantibody (E) or with anti-GFAP antibody (F). Although all cells stainedwith anti NF or anti-NSE antibodies, no cell staining was observed withanti-GFAP antibody (Negative magnification 33× (D,E); 66× (F)).

[0071] Calls began to proliferate by day 2 and newborn cells were smalland bipolar in shape. Short processes roughly equal in length to cellbodies started to emerge from parent cells. Over the next 2-3 days, 1 or2 of the processes started to grow rapidly and contacted the neighboringcells (FIG. 2A). By day 7, both the cell bodies and the processes hadincreased in size and an extensive interconnecting network of processeshad formed. This morphological progression resembled hippocampalpyramidal neuronal morphologies previously described in vitro (Bankerand Cowan, Brain Res., 126:397-425, 1977; Banker and Cowan, J. Comp.Neurol., 187:469-494, 1979). When cells growing in culture for 1-2 weekswere passaged, more of these cells had processes than did the-cellsnewly cultured from the brain (FIG. 2B). It is possible that many ofthese processes survived passaging, albeit partially amputated. Cellspassaged and kept in culture for 14 days in the presence of bFGF formedaggregates and were interconnected by an extensive network of processesforming a lattice-type pattern (FIG. 2C). Few cells divided in openareas; most cell division occurred in the aggregates.

[0072] The cultures were characterized by immunostaining or differentantigenic markers (FIGS. 2D, E, F; Table 1). All cell somata and theirprocesses immunostained strongly with an antibody against NF proteinwhich is specifically expressed by neurons (FIG. 2D). Similarly,anti-NSE antibody stained all cells in our culture (FIG. 2E; Table 1).The neuronal nature of the cells proliferating in response to bFGF wasfurther demonstrated by the binding of tetanus toxin, a specific markerfor neurons (Neale, et al., Soc. Neurosci. Abst, 14:547, 1988).NeuroTag™ green stained cell bodies and processes of all cells in theculture (FIG. 1C), indicating that the cells were neurons and that no orvery few non-neuronal cells were present in the cultures. The largeoptical depth of field with the objective used (10×) fails todemonstrate the localization of NeuroTag™ signal as membrane bound.

[0073] The cultures were tested by immunostaining for the presence ofnon-neuronal cells (Table 2). Lack of immunostaining with antibodiesagainst GFAP indicated the absence of astrocytes (FIG. 2F). In a controlexperiment anti-GFAP antibody (Amersham), at the same concentration(1:10,000) immunostained rat C6 and 9L and human U373 glioma cells. Theabsence of oligodendrocytes and fibroblasts in our cultures wasdemonstrated by the lack of staining for Gal C, vimentin or fibronectin(Table 2). As a control, rat C6, 9L and human U373 glioma cells werestained with vimentin (1:800) at the same concentration as used forneuronal cultures. The results of immunostaining for other antigenicmarkers are shown in Table 2: these data support the conclusion that thecultures consist of neurons uncontaminated by non-neuronal cells. TABLE2 PROPERTIES OF PRIMARY HIPPOCAMPAL NEURONS - ANTIGENIC MARKERS FORNEURONS AND NON-NEURONAL CELLS CULTURING CHARACTERISTICS SubstrateDependency Yes Basic FGF Dependency Yes Freeze-Thaw Viability YesANTIGENIC MARKERS CELL SPECIFICITY Neurofilament (NF) Neurons ++^(a)GFAP Glia −^(b) Nestin Stem cells ++ Vimentin Gliaprecursors/fibroblasts − NSE Neurons +^(c) OX-42 Microglia/macrophages −Galactocerebroside Oligodendrocytes − MAP2 Dendrites + Basic FGFreceptor Neurons/glia + Fibronectin Fibroblasts −

Example 4 Analysis of Perpetualized Neurons In Vitro

[0074] Analysis of primary neurons in culture at the ultrastructurallevel demonstrated the histotypic neuronal morphology of these cells(FIGS. 3 and 4), in agreement with previous ultrastructural studies(Bartlett and Banker, J. Neurosci., 4:19440-19453, 1984. Rothman andCowan, J. Comp. Neurol., 195:141-155, 1981; Peacock, et al., Brain Res,169:231-246, 1979). FIG. 3 shows transmission electron micrographs ofprimary neurons in culture. A shows a pyramidal-shaped primaryhippocampal neuron showing both the soma and processes, including amajor apical process (arrow) and a finer caliber process (arrowhead).Bar=10 μm. B shows an enlarged view of the neuronal soma shown in panelA. Bar=1 μm. C shows a portion of the major apical process of the neuronshown in panel A. This process is dominated by microtubules andpolysomal ribosomes identifying it as a primary dendrite. Bar=1 μm. Dshows contact between two neuritic processes. Bar=0.1 μm.

[0075] The well-differentiated neurons exhibited a histotypic pyramidalmorphology, including a primary, apical dendrite with multipleramifications, finer caliber axons, and characteristic nuclearmorphology (FIGS. 3 and 4). A TEM micrograph of a pyramidal-shapedprimary hippocampal neuron is shown in FIG. 3A. The level of thissection encompasses both the soma and processes, including a majorapical process (arrow) and a finer caliber process emerging from thebasal aspect of the soma (arrowhead). Other processes from adjacentneurons are also seen. The soma of the neuron has a euchromatic nucleuswith a peripheral rim of heterochromatin and a somewhat reticulatednucleolus (FIG. 38). Mitochondria and microtubules are present in theperikaryal cytoplasm, which is dominated by rosettes of polysomalribosomes. A portion of the major apical process of the neuron isdominated by microtubules and polysomal ribosomes identifying it as aprimary dendrite (FIG. 3C) Contact between 2 neuritic processes is shownin FIG. 3D. The larger process containing a mitochondrion, microtubulesand vesicles is being contacted by a swollen, bouton-ike structurearising from a finer caliber process. The junction between suchprocesses is typically vague and immature at this age in culture.Although the membranes at the site of contact appear to be uniformlyparallel, there is little indication of further assembly of synapticstructures. The contents of the bouton-like process ending are unclear,appearing to be an accumulation of vesicles, with a possible coatedvesicle near the site of contact.

[0076]FIG. 4 shows scanning electron micrographs of primary neurons inculture. A shows an overview of primary hippocampal neurons' in cultureincluding well-differentiated pyramidal somata (arrow) with largeprocesses containing multiple levels of branching andless-differentiated, rounded neurons with large, extended processes(arrowheads). Bar=50 μm. B shows a major apical dendrite emerging from awell-differentiated pyramidal neuron showing a smooth, regular caliberprocess just proximal to the first (major) bifurcation with severalsmaller processes, possibly axons emerging from it. The PORN/laminincoating the vessel surface can be seen as a porous carpeting which isabsent in some patches. Bar=2 μm. C shows a well-differentiated neuron(in the middle of the field) possessing a large pyramidal soma (compareto FIG. 3A) and a large apical dendrite (arrowheads) contacted by anumber of processes from other neurons. Other less-differentiatedneurons which are fixed in the process of dividing were also present(arrows). Bar 20 μm. D shows an enlarged view of the dividing neuron inthe upper field of view in panel C. The membrane connecting the twodaughter cell components is clearly continuous, although cytokinesis isapparently underway. Note the process extension from thisless-differentiated neuron, indicating some degree of differentiationduring mitosis. Bar=10 μm.

[0077] Scanning EM of primary hippocampal neurons in culture showed thediversity of morphologies present, with some well-differentiatedpyramidal somata (FIG. 4A; arrow) extending large processes which showmultiple levels of branching and some less-differentiated, roundedneurons. Even these rounded neurons possess large, extended processes(FIG. 4A; arrowheads). Closer examination of the major dendriticprocesses arising from the well differentiated neurons shows largecaliber processes with acute bifurcations (FIG. 48). A number of smallcaliber, axon-like processes are seen emerging from these major apicaldendrites (FIG. 4B). Well-differentiated neurons typically possess alarge pyramidal soma (FIG. 4C compare to FIG. 3A). Whenless-differentiated neurons are examined, many of these are found tohave been fixed in the process of dividing (FIG. 4C; arrows). A closerview of the dividing neuron shows that, although cytokinesis isapparently underway, the membrane connecting the two daughter cellcomponents is clearly continuous. The daughter cell component to theright is extending a fine caliber, possibly axonal, process into theforeground. Extending from this component into the upper right of thefield is another thicker, dendrite-like process which undergoes severallevels of branching.

[0078] In contrast to the previous, ultrastructural reports (Banker andCowen, Brain Res., 126.397-425, 1977; Banker and Cowen, J. Comp.Neurol., 187:469-494, 1979; 29 Rothman, et al., J. Comp. Neurol.,195:141-155, 1981), the perpetualized neurons had fine caliber axonalprocesses which emerged from the soma in a histotypic manner in additionto the dendritic origin (FIGS. 3A and 4D). These somatic axonalextensions may be the result of the high levels of trophic support.Less-differentiated neurons typically had rounder somata with fewer,less elaborate processes. Even rounded neurons, differentiatedadequately to extend processes, appeared capable of proliferating (FIG.4D). Neuronal processes and somata have been identified based on boththe ultrastructural surface morphology and organelle content, whichclearly demonstrates that both the well-differentiated andproliferating, less-differentiated cells are neurons.

Example 5

[0079] Effects of Different Growth Factors on Cell Culturing

[0080] Tissues were dissected from the specific areas of the centralnervous system (CNS) and dissociated as described in EXAMPLE 1. Aftercentrifugation, cells were resuspended in N2 medium and cells werecounted. Approximately 0.5-1.0×10⁶ cells were plated an PORN/laminincoated 24 well plates in N2 medium containing different growth factorsat different concentrations, depending on the specific region of theCNS. Cells were cultured at 37° C. in 5% CO₂. Cells were examined, andif necessary, counted in 5 separate areas in a well at day 1, 4, and 7to determine the growth rates in the presence of various growth factors(TABLE 3).

[0081] In some experiments, no proliferation of cells was observed inthe presence of certain growth factors. In some cases there was massivecell death, although a small population of cells survived up to day 4.These surviving cells did not look healthy, however, addition of bFGF at20-100 ng/ml (depending on the origin of the tissue), in N2 mediumrescued these surviving cells as evidenced by this proliferation (see a,TABLE 3). TABLE 3 EFFECTS OF DIFFERENT GROWTH FACTORS ON PROLIFERATIONOF CNS NEURONS Region Growth Factor Concentration Effect HippocampusbFGF 20 ng/ml ++ NGF^(a) 20 ng/ml − EGF 20 ng/ml + BDNF 20 ng/ml − NT3ND* + Septum bFGF 100 ng/ml  ++ NGF^(a) 100 ng/ml  − EGF^(a) 100 ng/ml − BDNF^(a) 100 ng/ml  − NT3^(a) ND* − Locus Ceruleus bFGF 50 ng/ml ++NT3 ND* − Ventral bFGF 50 ng/ml ++ Mecencephalon BDNF 50 ng/ml − EGF 50ng/ml − Cerebellum bFGF 20 ng/ml ++ EGF 20 ng/ml + NGF 20 ng/ml − BDNF50 ng/ml − NT3 50 ng/ml − Spinal Cord bFGF 20 ng/ml ++ NT3 20 ng/ml +

Example 6 Preparation of Adult Neuronal Cultures

[0082] Hippocampi of normal adult Fisher rats were dissociated and grownin serum-free culture containing bFGF as described in Example 1.Briefly, hippocampi were dissected from normal adult (>3 mo) rat brains.Most of the choroid plexus, ependymal lining and subependymal zone wasremoved. Cells were dissociated mechanically and enzymatically usingmethods described previously (Ray, et al., 1993, supra) with thefollowing modifications: After enzymatic dissociation in apapain-protease-DNase (PPD) solution (Hank's balanced salt solutionsupplemented with 4 mM MgSO₄ and 0.01% papain, 0.1% neutral protease and0.01% DNasel), cells were centrifuged at 1000 g for 3 min, resuspendedand triturated in 1 ml of DMEM:F12 (1:1) high glucose medium (IrvineScientific)+10% fetal bovine serum (10% FBS) (Sigma). Cells were platedonto uncoated plastic T-75 culture flasks (Costar) at 1×10⁶ viable cellsper flask in 10% FBS medium overnight. Lower cell densities were usedwith smaller culture flasks or Lab-Tek slide chambers (Nunc). Cells wereoccasionally plated onto cultureware previously coated withpolyomithine/laminin as described in Example 1. The medium was removedthe next morning and replaced with, serum-free medium: DMEM:F12÷N2(GIBCO) at 1 ml/100 ml medium (N2),+bFGF (recombinant human bFGF,Syntex/Synergen Consortium; (Ray, et al., supra) at 20 ng/ml. Flaskswere incubated 1-3 weeks, when half of the medium was removed andreplaced with the same volume of fresh N2+bFGr. Partial medium exchangewas made 1-2× weekly or as needed. Cultures were examined andphotographed using phase contrast microscopy (Nikon Diaphot).

[0083] In a number of experiments cells were harvested and transferreddirectly to new flasks or Lab-Tek slide chambers where they attachedimmediately and started proliferating, or occasionally passaged usingtrypsinization with ATV trypsin (Irvine Scientific), followed bywashing, centrifugation and re-plating in N2+bFGF.

[0084] Primary cultures of neurons from adult rat hippocampi werereplicated more than 15 times. To determine whether 10% FBS or N2 mediumcould account for the observed effects, some cultures were grown in 10%FBS or N2. Only cultures with bFGF developed large numbers of neurons.Some dissections were made of the CA1, CA3 and dentate gyrus regions.Neurons were generated from all three regions. Cultures are described inthree overlapping temporal stages: early, middle and late.

[0085] Early cultures (1-21 days) were characterized by cell attachmentto the substrate, cell proliferation and expression of mature neuronalfeatures. After clearing cell debris in the medium, single cells thatwere phase-bright and round and doublet cells, suggestive of celldivision, were observed at two days in vitro (d.i.v.). Numerousphase-bright cell bodies displayed processes tipped with growth cones.Cells of neuronal morphologies, i.e., phase-bright multipolar cell bodywith thin branching processes, were observed as early as 5 d.i.v.Processes developing complex branching patterns and evidence ofincomplete cytokinesis or potential synapse formation betweenpresumptive sibling neurons were observed as early as 8 d.i.v. (Nikonphase contrast-2 microscope/negative magnification 33×46×).

[0086] For SEM, cultures were fixed in 2% glutaraldehyde in 0.1 M PBS,osmicated in 1% aqueous osmium tetroxide, dehydrated in a graded ethanolseries, critical point dried with liquid carbon dioxide, attached tostubs with silver paste, sputter coated to 300 Å with gold/palladium andexamined and photographed in a Cambridge Scanning Electron Microscope(Stereoscan 360).

[0087] Examination of the three-dimensional morphology ofearly/intermediate stage cultures using scanning electron microscopy(SEM) revealed numerous cells of both neuronal and epithelioidphenotypes. Lacy neural networks were observed as with phase microscopy.Cells that appeared to be dividing were also observed. Highermagnification revealed that the processes between cells and cellaggregates interpreted at the light microscope level as a single processwere frequently 2 or more fasciculated processes.

[0088] Intermediate cultures (approximately 14-60 days) werecharacterized by increasing numbers of cells, the presence of neuralnetworks, the development of mature neurons and initial cell aggregateformation. Rudimentary networks of fine processes connecting small cellclusters were observed as early as 14 d.i.v. Networks of cellsdisplaying neuronal morphologies became more extensive and complex.Cells in the cultures were heterogeneous although individual patches ofneural networks displayed a uniform morphological phenotype. Individualcells away from clusters or networks also developed well differentiatedmorphological features characteristic of mature neurons, with largephase-bright multipolar-cell bodies and long thin processes thatbranched repeatedly. Processes of these cells often measured nearly 1000μm, and large indented nuclei and prominent nucleoli could be seen indifferent focal planes. Some neurons displayed small thorn-likeprojections indistinguishable from dendritic spines on processes.

[0089] Late cultures (approximately 2 to 7 months) were characterized byincreasing numbers of cells to confluence, increasing cell aggregatesconnected by processes and a background of individual cells. Whensubstrate space was available, cells with multiple thin processescharacteristic of earlier stages continued to be observed. Callaggregates were connected by cable-like neurites. Large numbers of cellaggregates developed and the entire substrate became covered with cellaggregates and individual cells that appeared to have migrated from thecell aggregates. While many background cells displayed features typicalof neurons, some cells expressed features typical of astrocytic glia

Example 7 Gene Expression in Cultured Neuronal Cells

[0090] The presence of NFh and GFAP was further confirmed by reversetranscriptase-polymerase chain reaction (RT-PCR) with RNA obtained fromcells harvested after different times in culture.

[0091] RNA was extracted using the guanidinium cesium chloride (CsCl)method (Current Protocols in Molecular Biology, Vol. 1, WileyInterscience, NY, F.M. Ausubel, et al., eds, 1988). The pellets weresolubilized in 1 ml solution D (4.0 M guinidine thiocyanate, 25 mM Nacitrate, 0.5% sarcosyl and DEPC treated H₂O) after thawing, trituratedgently and the cell lysate was transferred to CsCl previously pouredinto centrifuge tubes. The level of the CsCl was marked, and the tubeswere weighed and balanced. The tubes were centrifuged in a BeckmanUltracentrifuge overnight at 40,000 rpm at 20° C. The next morning,solution D was removed, and the interface washed with solution D. TheCsCl solution was carefully poured off, and the RNA pellet was rinsedwith 70% EtOH (made with DEPC water). After the pellet was dry it wassolubilized in DEPC-H₂O and the remainder was stored in EtOH at −70° C.

[0092] A RT-PCR method was used to obtain cDNA's (Ausubel, et al., 1988,supra). The reaction tube contained 4 μl RNA (10-100 ng), 8 μl(sufficient DEPC-H₂O to bring the volume up to 20 μl), 2 μl 10×PCRbuffer, 2 μl 10 mM d NTP's, 1 μl random hexamers, 3 μl 24 mM MgCl, 0.125μl AMV-RT and 0.5 μl RNasin. A drop of Nujol mineral oil was added toeach tube and the reaction was run in a Perkin Elmer Thermal Cycler: 42°C.—75 min; 95° C.—10 min; and held at 4° C.

[0093] A PCR method was used to further amplify the specific desiredcDNAs from the cDNAs obtained above. Each reaction tube contained 5 μlcDNA, 9.5 μl PCR buffer, 7.25 μl MgCl₂, 0.2 or 0.3 μl ³²P-dCTP, 1.5 μl10 mM dNTP's, 0.5 Taq polymerase, 6μ, 1 primers (2 μl [1 μl (F (Forwardrx):5′)+1 μl R (reverse:3′) each of RPL 27, NFh, GFAP, NGF or bFGF) andsufficient H₂O to bring the volume to 100 μl. The reacton was run in aPerkin Elmer Thermal Cycler: 94° C.—10 min and held at 4° C.

[0094] AmpliTaq DNA polymerase was from Perkin-Elmer, AMV reversetranscriptase, random oligonucleotide hexamer primers and recombinantRNasin ribonuclease inhibitor were from Promega, specific primers weremade to order. dNTP's were from New England Nuclear. The primers were asfollows:

[0095] NFh Forward (F) primer: 5′-GAGGAGATAACTGAGTACCG-3′ Reverse (A)primer: 5′-CCAAAGCCAATCCGACACTC-3′

[0096] GFAP F primer: 5′-ACCTCGGCACCCTGAGGCAG-3′ R primer:5′-CCAGCGAGTCAACCTTCCTC-3′

[0097] Gel electrophoresis of cDNA-samples obtained from PCRamplification was done on a 6% non-denaturing polyacrylamide gel. Somesamples and their corresponding digests were run on agarose gels usingethidium bromide to bind and illuminate the DNA under UV light A 123 bpmolecular ladder was run in a lane beside the samples. Electrophoresiswas done for varying periods of time, and the resulting gels were driedfor 1 hr on a gel drier. Autoradiographic films of dried acrlyamide gelswere developed for periods, of time ranging from several hours to 10days.

[0098] Relative levels of mRNA were analyzed quantitatively usingdensitometry over cDNA bands identified as NFh, GFAP, NGF and bFGF fromNorthern blots of cultures grown 36 to 117 days. A diverging pattern ofmRNA expression was apparent. Expression of message for NFh wasrelatively low. At about 2 months, the relative levels switched andexpression of mRNA for GFAP increased over time then droppeddramatically at about 4 months in culture, while expression of NFh fellover time, then rose slightly after 4 months in culture.

[0099] Digests of NFh were performed on samples remaining from earlierreactions using a cocktail consisting of 40 μl sample, 5 μl React #6buffer (50 mM Tris, pH 7.4, 6 mm MgCl₂, 50 mM KCl, 50 mM NaCl) and 5 μlPvu II restriction enzyme, and reacted for 1 hr at 37° C. The productswere run along with a 123 bp molecular ladder on a 6% acrylamide gel.The gel was dried, exposed on film for varying periods of time, and theresulting autoradiograms were examined for bands at the predictedmolecular weight levels. mRNA for both NFh and GFAP was present in allcultures at the times examined.

Example 8 Immunocytochemistry

[0100] To determine whether cells expressed antigens typical of neuraltissue, cultures were processed for immunocytochemistry. Cells werefixed for 30 min in 4% paraformaldehyde at room temperature or 37° C.,incubated with 0.6% H₂O₂ in TBS followed by incubation in blockingsolution. Me cultures were incubated with primary antibody atappropriate dilutions overnight at 4° C. The next day cells were rinsedwith diluent and incubated in secondary antibody for 1 hr at roomtemperature, rinsed with TBS and incubated in ABC solution (equalamounts of avidin and biotin) for 1 hr at room temperature. They wererinsed with TBS and incubated with DAB-NiCl for variable reaction times,rinsed with TBS, dried overnight, dehydrated through graded series ofalcohol and mounted in histoclear. Antibodies were from the followingsources and used at the dilutions indicated. Monoclonal antibodies: highmolecular weight subunit of neurofilament protein (NF-H 200 kD; 1:24);middle molecular weight subunit of neurofilament protein (NF-M 160:1:10); glial filament acidic protein (GFAP; 1:100) and synaptophysin(1:10) (Boehringer Mannheim); calbindin (Cal-b; 1:200) and microtubuleassociated protein 2 (MAP2; 1:500) (Sigma); neuron-specific enolase(NSE; 1:200) (DAKO). Polyclonal antibodies: NF-H 200 (1:250); NF-M 150(1:500); NF-L 68 (1:125); GFAP (1:1000) and gamma amino butyric acid(GABA; 1:200) (Chemicon); NSE (1:800) (Polysciences); galactocerebrocide(Gal-C; 1:5000). (Advanced Immuno Chem.); bFGF (1:1000); (WhittierInstitute, La Jolla, Calif.). Normal horse and goat serum, biotinylatedgoat anti-rabbit IgG, horse anti-mouse IgG and ABC Vectastain Elite kitwere from Vector Laboratories (Burlingame, Calif.). There was nodetectable staining when primary antibody was omitted and replaced withnon-immune serum.

[0101] Neuro-specific enolase (NSE)-positive cells were observed inearly cultures. By 170 d.i.v., a majority of the cells wereimmunoreactive for high molecular weight neurofilament protein (NFh, 200kD) that is characteristic for adult neurons, as well as the middle andlow molecular weight of NF. Most NF-positive cells also hadmorphological properties of neurons. A small subpopulation of cells(less than 10%) was immunopositive for calbindin, which is specific forgranule cells. Cells with neuronal phenotypes were also immunopositivefor MAP2 with reaction product localized to the cytoplasm of cell bodiesand proximal processes. Cells with astroglial morphology stained forGFAP Less than 1% of the cells strained for GABA. Many cells wereimmunopositive for bFGF and a few small round cells were immunopositivefor galactocerebroside.

Example 9 Characterization of Neuronal Cell Growth In Vitro

[0102] To determine whether cells were proliferating and, if so, assessthe nature of such cell types, cultures were incubated inbromodeoxyuridine (BrdU) for 36 hours and then dual labeled forimmunofluorescence with BrdU and neuron-specific enolase (NSE) or glialfilament acidic protein (GFAP).

[0103] For BrdU incorporation, cells were cultured in glass Lab-Tekslide chambers for 11 days. The medium was replaced with fresh N2+bFGFcontaining bromodeoxyuridine (BrdU) labeling reagent (1 μl/ml medium;Amersham) and the cultures were incubated an additional 1 or 4 days fora total of 12 or 15 days. Cells were fixed in 4% paraformaldehyde inphosphate-buffered saline (PBS) for 30 min, washed with PBS, blockedwith 10% normal donkey serum (Jackson ImmunoResearch Labs) in PBS, andreacted with monoclonal antibody to BrdU (BrdU; undiluted; Amersham)followed by donkey anti-mouse IgG coupled to Cy-5 (JacksonImmunoResearch Labs). Some cultures were dual labeled with polyclonalantibody against neuron specific enolase (NSE; 1:800) or antibodyagainst glial fibriliary acidic protein (GFAP; 1:1000). Secondaryantibody for the polyclonals was donkey ant-rabbit IgG conjugated tofluorescein isothiocyanate (FITC; Jackson ImmunoResearch Labs). Slideswere mounted in Slow-Fade mounting reagent (Molecular Probes). Cellswere visualized using a BioRad MRC 600 Confocal Scanning LaserMicroscope. Images were collected and transferred to an Apple MacintoshQuadra 700, merged using Adobe Photoshop 101 and printed out on a GCCfilm recorder. Confocal scanning microscopy revealed cellsimmunoreactive for NSE and BrdU, as well as BrdU and GFAP positive cellsshowing that cells expressing neuronal and glial cell markers dividingin these cultures.

[0104] To determine if cell numbers in culture were increasing, cellswere counted over a 2 week period in 10 random fields. Thirty-sevenpercent of cells originally attached had survived by the second day inculture. Within 5 days, cell numbers had risen to slightly above theiroriginal level, and by the end of seven days, there were nearly twice asmany cells. By the end of 2 weeks, there were almost five times as manycells as on the first day in culture.

[0105] The most important result of this study is the demonstration ofneuronal proliferation from normal adult hippocampus when cultured withbFGF. Neurons survive and proliferate abundantly for long periods oftime, more than 200 d.i.v. to date; this is the first suchdemonstration.

[0106] It has been reported that initiation of cell division of isolatedadult brain (striatal) cells in culture requires epidermal growth factor(EGF), but not bFGF at 20 ng/ml, and a non-adhesive substrate (Reynolds& Weiss, Science, 755:1707, 1992). In contrast, the present datasupports that: 1) bFGF at 20 ng/ml acts as a strong mitogen and as asurvival factor in adult as well as fetal hippocampal cultures; 2)proliferation occurs in substrate-bound cells and aggregates, i.e.,cells not in suspension, and 3) many, if not most, of the cells thatattach are bFGF-responsive.

[0107] Limited neuronal division as been reported over short times inother culture systems of adult brain (Reynolds, supra; Richards, et al.Proc. Natl. Acad. Sci, USA, 89:8591, 1992). In the present studyproliferation was confirmed by BrdU incorporation and the finding thatcell numbers increased almost 500% over a 2 week period. Although it hasbeen reported that less that 1% of adult striatal cells initially platedproliferate (Reynolds, et al. supra), in the cultures described hereinnearly 40% of cells which initially attached survived to the second dayin culture, suggesting that many cells are present in adult hippocampusthat have the capacity to proliferate.

[0108] Evidence from several independent experiments supports the ideathat most cells in these cultures not only are neurons, but they aremature neurons which express morphological, biochemical and molecularfeatures characteristic of adult neurons. While glia are also generated,glia were a minority phenotype in most cultures. Similar findings havebeen reported for fetal rat hippocampus neurons cultured with bFGF (Ray,et al., 1993, supra).

[0109] The source of the proliferating neurons for the adult brainremains to be determined. The cells could be mature functioning neuronsthat were saved and induced to proliferated by high concentration ofbFGF; the cells could be stem cells of suspected proliferation zones; orthe cells could be partially committed neurons (neuroblasts) that havebecome quiescent due to a reduction in high levels of bFGF present onlyin the embryonic brain and/or because of contact inhibition. While it isunlikely that all neurons that were observed and generated could beaccounted for the mature neurons saved following plating, work is inprogress to determine whether mature differentiated neurons are capableof in vitro survival and proliferation through dedifferentiation. It ispossible that the subventricular zone (SVZ) could be the source of thesecells, since SVZ of mammalian forebrain has been shown to be the sourceof these cells that differentiate into neurons and glia in adult mice(Clois, et al., Proc. Nat'l Acad. Sci. U.S.A., 79:2074, 1993). However,it is not likely that the SVZ could have served as the main source ofproliferating cells in the cultures of the invention, since theependymal lining/SVZ, along with choroid plexus, was stripped away.These neurons could be derived from a small population of embryonic stemcells that survives in the adult brain in a dormant, non-proliferativestate, as has been suggested exists in adult mouse striatum (Reynolds,et al., supra). Alternatively, these neurons could be neuronal precursorcells existing in adult mammalian brain that require discrete epigeneticsignals for their proliferation and differentiation as has beenspeculated for adult mouse brain (Richards, et al., supra).

[0110] In addition to stem cells of the SVZ, there is a large populationof neuroblast in the normal adult mammalian hippocampus that can beinduced to generate large numbers of neurons over long periods of timeunder appropriate in vitro conditions. It is possible that this couldalso be true in vivo, a concept that has profound implications for basicand clinical neuroscience. This could mean that normal hippocampus and,by extension, normal CNS has a reservoir of cells that can be activatedunder appropriate conditions to replicate large numbers of neurons.Thus, neuroblasts could be present not only in cultures of fetal CNS,but also in cultures of adult CNS and in the adult CNS in situ.

Example 10 Long-Term Culture of Neurons from Adult Hippocampus

[0111] Brains of adult Fisher rats (>3 months old) were dissected, themenengies removed, and the hippocampai dissected out. The tissues weretransferred to a 15 ml tissue culture tube and washed three times with 5ml Dulbecco's phosphate buffered saline (D-PBS). After the last wash,the tissue was pelleted by centrifugation at 1000 g for 3 min and thewash solution removed The tissue was suspended in 5 ml papain-neutralprotease-DNase (PPD) solution and incubated at 37° C. for 20-30 min withoccasional shaking. The solution was made in Hank's balanced saltsolution supplemented with 12.4 mM MgSO₄ containing 0.01% papain, 0.1%neutral protease and 0.01% DNase I (London, R. M. and Robbins, R. J.,Method. Enzymol., 124:412-424, 1986).

[0112] Hippocampai were mechanically dissociated by tituration with amedium bore pasteur pipet (about 20 times). Cells were pelleted bycentrifugation at 1000 g for 3 min. The cells were resuspended in 1 mlDMEM:F12 (1:1) medium containing 10% fetal bovine serum, 3.9 mMglutamine (complete medium). Cell clumps were mechanically dissociatedby tituration with medium to fine bore pasteur pipets (about 20 timeswith each). Cells were washed with 5-10 ml complete medium twice bycentrifugation. Cells were taken up in 1 ml complete medium, dissociatedby tituration and counted in a hemocytometer. Cells were plated at adensity of 1×10⁶ cells/T75 flasks (Coaster) and incubated at 37° C. in5% CO₂/95% air incubator. After incubation for 18-24 hours, the mediumwas changed with N2 medium (1:1 mixture of DMEM/F-12 containing 20 nMprogesterone, 30 nM sodium selenite, 100 μM putrescine, 3.9 mMglutamine, insulin (5 μg/ml) and transferrin (100 μg/ml)] containing 20ng/ml FGF-2 (bFGF). To date cells have been cultured for at least 7months and have been cultured from 15 different independent dissections.

[0113] The neuronal nature of cells were determined by examination ofmorphology at light and scanning microscope levels. Immunocytochemicalanalysis showed that these cells expressed neuron-specific enolase,neurofilament medium and high molecular weight proteins, MAP-2, andcalbindin (only a small population). Some cells in these cultures alsostained for GFAP indicating the presence of astrocytes in thesecultures. The proliferation of adult neuronal cells in cultures wasdetermined by bromodeoxyuridine (BrdU) incorporation. The nuclei ofcells expressing neuron-specific enolase were immunostained with anantibody against BrdU indicating cell proliferation in culture.

Example 11 In Vivo Survival of Perpetual Hippocampal Neurons AfterGrafting in the Adult Brain

[0114] Embryonic hippocampal neurons were cultured in N2 mediumcontaining 20 ng/ml bFGF. Cells were passaged and allowed to grow until7080% confluent. The medium was replaced with fresh medium (N2+bFGF)containing ³H-thymidine (1 μCi/ml; specific activity: 25 Ci/mmol) andallowed to grow for 3.5 days. Cells were harvested from flasks bytrypsinization and washed with D-PBS 3 times by centrifugation. Cellswere resuspended in 2 mls of D-PBS containing 20 ng/ml bFGF, dissociatedby tituration and counted in a hemocytometer. After centrifugation toremove the supernatant, cells were resuspended at a concentration of60,000 cells/μl. One microliter of cell suspension was injected in thehippocampus of adult Fisher rats (>3 months old). Animals were perfusedwith 4% paraformaldehyde, and the brains removed. Brain sections weretreated with antibodies with calbindin, GFAP and NF-H proteins. Thesections were dipped in emulsion and developed after 6 weeks. Number ofcells with at least 12 grains on them were counted in every 12 sectionsfor each animal (3 animal total). At three weeks, an average of 17%cells implanted in the brain survived (Table 4). TABLE 4 SURVIVAL OFPERPETUAL HIPPOCAMPAL NEURONS IN ADULT RAT HIPPOCAMPUS AVERAGE # CELLSANIMAL WITH GRAINS/SECTION % CELLS SURVIVING 1 222 15 2 233 19 3 244 17

[0115] Although the invention has been described with reference to thepresently preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention.

1 4 1 20 DNA Artificial Sequence Forward primer for PCR 1 gaggagataactgagtaccg 20 2 20 DNA Artificial Sequence Reverse primer for PCR 2ccaaagccaa tccgacactc 20 3 20 DNA Artificial Sequence Forward primer forPCR 3 acctcggcac cctgaggcag 20 4 20 DNA Artificial Sequence Reverseprimer for PCR 4 ccagcgactc aaccttcctc 20

1. A method of producing a neuroblast in vitro, the method comprisingculturing a neuronal cell in a vessel in a serum-free basal mediasupplemented with at least one trophic factor wherein a surface in thevessel allows attachment of the neuronal cell.
 2. The method of claim 1,wherein the neuronal cell is derived from neural tissue selected fromthe group consisting of hippocampus, cerebellum, spinal cord, cortex,striatum, basal forebrain, ventral mesencephalon, and locus ceruleus. 3.The method of claim 1, wherein the trophic factor is selected from thegroup consisting of nerve growth factor, brain derived neurotrophicfactor, neurotrophin, fibroblast growth factor, platelet derived growthfactor, epidermal growth factor, insulin growth factor, and transforminggrowth factor.
 4. The method of claim 3, wherein the fibroblast growthfactor is basic fibroblast growth factor.
 5. The method of claim 3,wherein the neurotrophin is neurotrophin-3.
 6. The method of claim 1,wherein the surface in the vessel is treated with a polybasic amino acidto allow attachment of the neuronal cell.
 7. The method of claim 6,wherein the polybasic amino acid is polyornithine.
 8. The method ofclaim 1, wherein the surface in the vessel is treated with anextracellular matrix molecule to allow attachment of the neuronal cell.9. The method of claim 8, wherein the extracellular matrix molecule isselected from the group consisting of laminin, collagen and fibronectin.10. The method of claim 1, wherein the neuronal cell is cultured inserum-containing media prior to culture in serum-free media.
 11. Amethod for identifying a composition which affects a neuroblast whichcomprises: (a) incubating components comprising the composition and theneuroblast, wherein the incubating is carried out under conditionssufficient to allow the components to interact; and (b) measuring theeffect on the neuroblast caused by the composition.
 12. The method ofclaim 11, wherein the effect is inhibition of the neuroblast.
 13. Themethod of claim 11, wherein the effect is stimulation of the neuroblast.14. The method of claim 11, wherein the neuroblast is derived fromneural tissue selected from the group consisting of hippocampus,cerebellum, spinal cord, cortex, striatum, basal forebrain, ventralmesencephalon, and locus ceruleus.
 15. The method of claim 11, whereinthe neuroblast is immortalized.
 16. The method of claim 15, wherein theneuroblast is immortalized by the introduction to the neuroblast of atleast one oncogene.
 17. The method of claim 15, wherein the oncogene isselected from the group consisting of v-myc, SV40 large T antigen andadenovirus E1A.
 18. The method of claim 11, wherein the neuroblastfurther comprises at least one exogenous gene.
 19. The method of claim18, wherein the exogenous gene encodes a receptor.
 20. The method ofclaim 19, wherein the receptor is selected from the group consisting ofreceptors which bind adrenaline, noradrenaline, glutamate, serotonin,dopamine, GABA, and acetylcholine.
 21. A culture system useful for theproduction and maintenance of a neuroblast comprising: (a) a serum-freebasal media containing at least one trophic factor; and (b) a vessel,wherein a surface in the vessel allows attachment of the neuroblast. 22.The culture system of claim 21, wherein the neuroblast is derived fromneural issue selected from the group consisting of hippocampus,cerebellum, spinal cord, cortex, striatum, basal forebrain, ventralmesencephalon, and locus ceruleus.
 23. The culture system of claim 21,wherein the trophic factor is basic fibroblast growth factor.
 24. Theculture system of claim 21, wherein the trophic factor is present at aconcentration of from about 1 ng/ml to about 100 ng/ml.
 25. The culturesystem of claim 21, wherein the trophic factor is present at aconcentration of from about 5 ng/ml to about 70 ng/ml.
 26. The culturesystem of claim 21, wherein the trophic factor is present at aconcentration from about 15 ng/ml to about 60 ng/ml.
 27. The culturesystem of claim 21, wherein the glucose is present at a concentrationfrom about 0.01% to about 1.5%.
 28. The culture system of claim 21,wherein the glucose is present at a concentration from about 0.1% toabout 0.6%.
 29. The culture system of claim 21, wherein the surface inthe vessel is treated with a polybasic amino acid.
 30. The culturesystem of claim 29, wherein the polybasic amino acid is polyomithine.31. The culture system of claim 21, wherein the surface in the vessel istreated with an extracellular matrix molecule.
 32. The culture system ofclaim 31, wherein the extracellular matrix molecule is selected from thegroup consisting of laminin, collagen, and fibronectin.
 33. A method oftreating a subject with a neuronal cell disorder comprisingadministering to the subject a therapeutically effective amount ofneuroblast.
 34. The method of claim 33, wherein the neuroblast containsan exogenous gene.
 35. The method of claim 34, wherein the exogenousgene encodes an oncogene.
 36. The method of claim 35, wherein theoncogene is selected from the group consisting of v-myc, SV40 large Tantigen and adenovirus E1A.
 37. The method of claim 34, wherein theexogenous gene encodes a receptor.
 38. The method of claim 37, whereinthe receptor is selected from the group consisting of receptors whichbind adrenaline, noradrenaline, glutamate, serotonin, dopamine, GABA,and acetylcholine receptor.
 39. The method of claim 34, wherein theexogenous gene encodes a ligand.
 40. The method of claim 39, wherein theligand is selected from the group consisting of adrenaline,noradrenaline, glutamate, dopamine, acetylcholine, gamma-aminobutyricacid, and serotonin.
 41. The method of claim 33, wherein the neuronaldisorder is selected from the group consisting of Alzheimer's disease,Parkinson's disease, Huntington's disease, stroke, and spinal corddamage.
 42. A cellular composition comprising an enriched population ofneuroblast cells.
 43. The composition of claim 42, wherein theneuroblast is derived from neural tissue selected from the groupconsisting of hippocampus, cerebellum, spinal cord, cortex, striatum,basal forebrain, ventral mesencephalon, and locus ceruleus.
 44. Thecomposition of claim 42, wherein the neuroblast is immortalized.
 45. Thecomposition of claim 44, wherein immortalization is achieved by theintroduction to the cell of at least one ancogene.
 46. The compositionof claim 45, wherein the oncogene is selected from the group consistingof v-myc, SV40 large T antigen and adenovirus E1A.
 47. The compositionof claim 42, wherein the neuroblast further comprises at least oneexogenous gene.
 48. The composition of claim 47, wherein the, exogenousgene encodes a receptor.
 49. The composition of claim 48, wherein thereceptor is selected from the group consisting of receptors which bindadrenaline, noradrenaline, glutamate, serotonin, dopamine, GABA, andacetylcholine.
 50. The composition of claim 47, wherein the exogenousgene encodes a ligand.
 51. The composition of claim 50, wherein theligand is selected from the group consisting of adrenaline,noradrenaline, glutamate, dopamine, acetylcholine, gamma-aminobutyricacid, and serotonin.